Self-aggregating protein compostions and use as sealants

ABSTRACT

An implantable member for use in the body is provided herein. This implantable member includes a porous biocompatible substrate; the substrate having at least one surface sealed fluid-tight with self-aggregating protein particles of substantially the same diameter range. The self-aggregated protein particles are formed from a deposited aqueous slurry of the protein particles.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. application Ser. No.09/877,867, filed Jun. 8, 2001, which is a continuation of U.S.application Ser. No. 09/292,688, filed Apr. 15, 1999, now U.S. Pat. No.6,299,639, which is a divisional of U.S. application Ser. No.08/814,533, filed Mar. 10, 1997, now U.S. Pat. No. 6,177,609, the entirecontents all of which are incorporated herein by reference.

FIELD OF INVENTION

The present invention relates to improved bio-compatible fluid-tightbarriers and barrier compositions for implantable articles. Moreparticularly, this invention relates to highly controllable,bio-compatible aqueous slurry compositions and to processes for formingfluid-tight barriers when these compositions are coated onto orimpregnated into an implantable prosthesis, such as a vascular graft orendoprosthesis.

BACKGROUND OF INVENTION

The use of implantable articles, such as porous synthetic vasculargrafts, is a well accepted practice in the art. To improve certainproperties of an implantable article, it is known to coat one or moresurfaces of such articles with bio-compatible compositions. Thesecoating compositions serve many different functions. For example, suchcoatings may render porous implantable articles blood-tight. Inparticular, U.S. Pat. No. 4,842,575 to Hoffman, Jr. et al. describes aprocess for rendering a synthetic vascular graft blood-tight bymassaging a collagen preparation into the porous structure of the graft.

Alternatively, such coatings may be used to deliver certainpharmaceutical agents to targeted areas on an implantable article. Forexample, U.S. Pat. No. 5,290,271 to Jernberg describes encapsulatedchemotherapeutics dispersed within a fluid or gel which are applied to asurface of an implant. In this way, the chemotherapeutic agents arereleased overtime to targeted areas on the implant.

Moreover, it is well known in the art to combine antibiotics,anti-thrombogenic agents and the like into coating and/or impregnatingcompositions that are applied to implantable articles. Such coatingsincrease the bio-compatibility of the implantable article by, forexample, decreasing the risk of infection and blood clot formationthereon.

Coating and impregnation compositions for implantable articles likethose described hereinabove can be made from a variety of materials.Such materials include, for example, biological molecule-containingcompositions, polymer-containing compositions and hybridpolymer-biological molecule-containing compositions. For example,coating compositions known in the art for implantable articles includesegmented linear polymers (U.S. Pat. No. 3,804,812 to Koroscil),heparinized polyurethane (U.S. Pat. No. 3,766,104 to Bonin et al.) andblock copolymers of polysiloxane and polyurethane (U.S. Pat. No.3,562,352 to Nyilas). Such compositions, however, may contain unreactedfunctional groups which participate in undesirable side reactions invivo and can inhibit cell ingrowth into, for example, a vascular graft.Such complications can lead to thrombus formation, infection, etc., atthe implantation site.

Biological molecule-containing coatings include, for example, suchextracellular matrix proteins as collagen, fibronectin, laminin andhyaluronic acid. The use of a slurry composition containing collagen toreduce the porosity of porous textile grafts is described in U.S. Pat.Nos. 4,842,575 and 5,108,424 to Hoffinan et al. both of which are herebyincorporated by reference. During the processing of such prior artslurries, collagen of appropriate size and purity was obtained frompreviously processed calf skins that were passed through a meat grinderand extruded through a series of filter sieves of constantly decreasingmesh size. A plasticizer was then added to the collagen slurry and thecomposition was applied to, e.g., the surface of a porous vasculargraft. The composition was then cross-linked and dried. The use of suchslurries provides an implantable article, such as a vascular graft, withacceptable bio-compatibility and blood-tightness.

Room temperature grinding of, for example, bovine hides as a step inproviding an aqueous dispersion of collagen is also described in U.S.Pat. No. 4,097,234 to Sohde et al. This patent, however, also teachesthat when the pH of, for example, a preparation of bovine hides ortendons is in the range where the collagen to be isolated is easilysolubilized or “swelled,” the collagen fibers can become nonuniform anddegraded due to the heat of friction caused from violent stirring ormechanical crushing of the preparation. Thus, Sohde et al. describemincing bovine corium and then milling it in two successive steps atabout room temperature, i.e., between 20° C.-25° C. The resultantaqueous dispersion is claimed to have collagen fibers of 4-12 μm indiameter, 2-25 mm in length and a viscosity of between 1/5 to 1/20 thatof similar prior art compositions. The end products of the Sohde et al.method include non-woven fabric, films, membranes, tubes or sheets foruse as artificial blood vessels, and sutures.

The method described by Sohde et al., however, suffers from the drawbackthat the grinding of the bovine tendons or hides is carried out at roomtemperature. Grinding of these tissues at room temperature raises thetemperature of the micro-environment at the grinding site and causes thecollagen to denature. This produces collagen having a higher solubilityboth in the medium in which it is produced and in the blood stream. Suchhigher solubility leads to premature absorption of the coating and cancause a deleterious affect on tissue ingrowth dynamics. Thus, thehealing characteristics of the device are substantially hindered.Collagen derived from such a process is clearly not desirable as asealant for an implantable article, such as for example, a porousvascular graft due to the risk of uneven or non-uniform distribution ofthe collagen particles within the sealant composition. Furthermore, thepremature absorption of the collagen coating can result in undesirableleakage of blood from, e.g., a sealant coated porous vascular graft.

As an alternate method for preparing implantable collagen, severalreferences describe cryogenic grinding of collagen. For example, U.S.Pat. No. 5,256,140 to Fallick describes a method for preparing anautologous source of injectable collagen for use in leveling skin havingdepressions therein. In this method, the skin of a patient who is toreceive the collagen composition is made brittle by cooling it tobetween −10° F. to 100° F. (−3.8° C. to −37.8° C.) using, for example,liquid nitrogen. The brittle skin is then crushed using a mortar andpestle or cryogenically ground using a freezer mill. This preparation isthen denatured and extracted in a weak acid solution so as to obtaindenatured collagen for delivery into a patient.

Similarly, U.S. Pat. No. 5,332,802 to Kehnan et al. describesauto-implantable collagen for use in plastic and ophthahnic surgery. Inparticular, to obtain the desired collagen preparation, a sample of apatient's skin is blended or homogenized by pulverizing the skin in afrozen state, such as by freezing the skin in liquid nitrogen andgrinding the frozen skin using a mortar and pestle or by way of acryopulverization mill. Such a treatment is used to increase thesolubility of the contaminates therein and to reduce the overallprocessing time of the preparation.

Such cryogenic methods, however, are directed to cosmetic surgery-typeapplications and are unsuitable for sealant compositions used inconjunction with porous implantable articles. In particular, suchmethods are directed to the small-scale preparation of injectablecollagen. Moreover, these compositions and methods are insufficient toproduce non-denatured, uniform sized collagen preparations having highlycontrolled viscosity ranges.

As previously stated, collagen has been widely used as a coating andimpregnating composition. In particular, its use as a fluid-tightbarrier for textile prostheses, such as vascular and endovascular graftshas been very successful. Processing of collagen, however, has manydifficulties, due to its inherent properties. For example, to make areproducible collagen slurry requires certain consistencies in the rawmaterial itself, as well as, the process steps and parameters. Naturallyoccurring materials such as collagen, will of course have many inherentvariations. In order to produce acceptable sealant compositions, thesevariations must be minimized. One way to do so is through controlledsourcing and processing conditions.

Notwithstanding such efforts to produce reliable and consistentcompositions which are able to form reproducible sealants for poroussubstrates, such as vascular grafts, other difficulties are presentwhich tend to compromise the quality and/or reproducibility of suchsealants. For example, it is well known that collagen denatures above acertain temperature, e.g. 37° C. Once denaturization occurs, there is aloss in its natural self-aggregating properties. As a result,crosslinking is preferred or required. Additionally, grinding of rawcollagen to specific particle sizes causes localized heating above itsdenaturization temperature. Such denaturization may go unnoticed in theearly processing stages and end up in the final product. Thus,conventional grinding methods have limited usefulness due to theexposure of, e.g., collagen, to excessive heat build-up caused by thefrictional grinding forces.

The prior art has also taught that cross-linking of the collagen was animportant step in forming an effective sealant composition. See, forexample, U.S. Pat. Nos. 4,842,575 and 5,108,424 to Hoffinan et al.described hereinabove. It has recently been discovered in the course ofthe present invention that by eliminating the potential fordenaturization and by controlling particle size, collagen compositionscan be made which, under specified viscosity ranges, form reproducible,high quality sealants. The specified particle size is obtained withoutthe concern for denaturization due to the use of cryogenic techniques asapplied to the communication process. The homogeneous particle sizepromotes uniformity in coating, further enhances the self-aggregatingproperties of the collagen and promotes the formation of a fluid-tightbarrier. As a result of the present inventive processes, effective, highquality fluid-tight barriers can be obtained without cross-linking ofthe collagen.

In summary, all of the above-cited references generally suffer from aninability to produce highly controllable and reproducible collagencompositions. Thus, there is a need for improved bio-compatible aqueousslurry compositions and processes for forming fluid-tight barriers onimplantable articles. In particular, there is a need for improvedcollagen compositions which contain non-denatured collagen having auniform particle size and which have highly controllable andreproducible viscosities. The present invention is directed to meetingthese and other needs.

SUMMARY OF INVENTION

The present invention provides an implantable member for use in thebody. This implantable member includes a porous biocompatible substrate;the substrate having at least one surface sealed fluid-tight withself-aggregating protein particles of substantially the same diameterrange. As described herein, “fluid tight” is intended to mean that theporous implantable member is rendered essentially non-permeable toliquids, such as for example, blood. The self-aggregating proteinparticles are formed from a deposited aqueous slurry of the proteinparticles. In some embodiments, the deposited slurry includes abiocompatible plasticizer. Moreover, in some embodiments, the depositedslurry includes a bio-active agent. Furthermore, in some embodiments,the deposited slurry includes a permeability lowering agent.

DETAILED DESCRIPTION OF THE INVENTION:

While this invention is satisfied by embodiments in many differentforms, there will be described herein in detail preferred embodiments ofthe invention with the understanding that the present disclosure is tobe considered as exemplary of the principles of the invention and is notintended to limit the invention to the embodiments illustrated anddescribed.

In accordance with the present invention, novel fluid-tight barriercompositions are provided. More particularly, novel compositions andprocesses are provided for the manufacture of collagen sealantcompositions for rendering porous implantable substrates blood-tight.

In one embodiment of the present invention, there is provided abio-compatible aqueous slurry for forming a fluid-tight barrier on asurface of a porous implantable article. For purposes of the presentinvention, “aqueous slurry” is intended to mean a water-basedcomposition which is sufficiently fluid to flow and contains a mixtureof finely divided particles including one or more self-aggregatingproteins. This aqueous slurry forms fluid-tight barriers when broughtinto contact with a surface of an implantable article.

For purposes of the present invention, “porous implantable article”includes any biocompatible article or substrate surface thereof to beimplanted within a body, and particularly refers to porous tubularprostheses. Preferably, the implantable article is a polymeric vascularprostheses, such as a knitted or woven polyethylene terephthalate (PET),polytetrafluoroethylene (PTFE) or polyurethane vascular graft orendovascular graft. These articles may be fabricated using knownmanufacturing techniques and materials. The vascular grafts of thepresent invention may be made from biologically compatible fibers oryarns, such as for example, polyethylene terephthalate, commonly soldunder the trademark DACRON and PTFE, or they may be made by knownextrusion and expansion techniques such as those used in manufacturingPTFE and polyurethane grafts. Dipping shaped mandrels in variouspolymers, such as polyurethane, are also useful. Furthermore, the graftsmay be knitted or woven and may be of a monofilament or a multi-filamentyarn. The term “vascular prostheses” will be used herein to include allgraft types, as well as, endoprosthesis, graft/stent andendovascular/stent combinations, mesh and hernia plugs and patches.

The bio-compatible slurry of the present invention includes aself-aggregating protein comminutate having a substantially uniformparticle size, a bio-compatible plasticizer and water. For purposes ofthe present invention, the term “self-aggregating protein” is meant toencompass any protein which, when in an aqueous solution, is able toself-associate. Such proteins are also selected based on their abilityto be absorbed by the body over time, to encourage healing and topromote tissue ingrowth into the implantable articles of the presentinvention. Suitable self-aggregating proteins include, withoutlimitation, many members of the extracellular matrix family of proteins,such as collagen, fibronectin, vitronectin, proteoglycan, laminin,hyaluronic acid, tenascin, integrin cadherin, and mixtures thereof.These self-aggregating proteins may be obtained from any suitablemammalian species.

Preferably, the self-aggregating protein of the present inventionbelongs to the collagen family of extracellular matrix molecules whichcurrently contains about 15 members. In addition to beingself-aggregating, collagens are also capable of self-assembly fromprocollagen molecules to collagen molecules to collagen fibrils. Usefultypes of collagens include collagen types I through XV. See e.g., BruceAlberts, Dennis Bray, Julian Lewis, Martin Raff, Keith Roberts and JamesD. Watson Molecular Biology of the Cell, 3rd ed. pgs. 963-1,000 (1994)which are hereby incorporated by reference. Preferably, theself-aggregating protein of the present invention is bovine type Icollagen.

For purposes of the present invention, the self-aggregating protein maybe present in the slurry composition at a concentration which allows theslurry to be easily and uniformly applied to an implantable article andwhich is sufficient to cause a fluid-tight barrier to form thereon.Preferably, the self-aggregating protein is present in the slurrycomposition at a concentration of from about 1% to about 3% by weight.More preferably, the self-aggregating protein is present in the slurrycomposition at a concentration of about 1.1% to about 2.0% by weight.

By “comminutate” it is meant that the self-aggregating protein isreduced to a powder of substantially uniform size by, for example,attrition, impact, crushing, grinding, abrasion, milling, chemicalmethods and combinations thereof followed by or including screening orsieving through the desired mesh size. Other techniques capable ofproducing particulates are also contemplated. Prior to comminutation,the self-aggregating protein is cryogenically processed to form a solidfrom the aqueous paste. The solid is then comminuted to the desiredparticle size. Frictional forces which contribute to localized heatbuild-up and denaturization do not deleteriously affect thecryogenically solidified mass. Thus, the particle size of theself-aggregating protein can be controlled without concern for loss ofits inherent self-aggregating properties which normally occur toproteins, such as collagen, when denatured. This is an importantfeature, because the smaller particle sizes required in the presentinvention may require rigorous comminution which would otherwise lead todenaturization.

Preferably, the self-aggregating protein is reduced to a uniformparticle size by a cryogenic milling process. As discussed in moredetail hereinbelow, this cryogenic milling process is accomplished bytaking a raw paste containing the self-aggregating protein and extrudingit through, for example a meat grinder to obtain a more convenient andworkable size, freezing the extruded material at cryogenic temperatures,maintaining such material at cryogenic temperatures while grinding thematerial to a powder, passing the powder through a sieve and collectingthe uniformly sized particles therefrom. The passage through ameat-grinder or similar machine does not generate sufficient heat toaffect the protein structure. For purposes of the present invention,cryogenic freezing means an almost instantaneous freezing by immersionin liquid nitrogen. Other suitable freezing techniques as known in theart are also contemplated so long as the self-aggregating protein ismaintained at a temperature below its denaturization point duringcomminution to the desired particle size.

By use of the cryogenic milling process of the present invention, afrozen self-aggregating protein paste is reduced to a powder of uniformparticle size without risk of denaturing the protein. In particular,because the protein is continuously maintained at cryogenic temperaturesthroughout the milling process, there is no risk that it will besubjected to denaturing temperatures, i.e., temperatures in excess of37° C., even at the localized milling surfaces. Thus, subsequent sealantcompositions formed from a protein processed in this manner are easilycontrollable, comprise particles which are highly uniform in diameter,and possess barrier properties which are readily reproducible.

For purposes of the present invention, “uniform particle size” meansthat the cryogenically milled and sieved particles derived from theself-aggregating protein have substantially the same diameter.Furthermore, the screen sizes for sieving the present cryogenicallymilled self-aggregating proteins are in the range from about 0.020inches to about 0.062 inches (0.5 mm-1.55 mm). A single screen size isgenerally chosen for a particular coating composition such thatuniformity in particle size exists in the final coating. This range ofscreen sizes produces self-aggregating protein particles havingdiameters of about 5 μm to about 750 μm, depending upon the chosenscreen size. Thus, self-aggregating particles have diameters in thisrange are suitable for forming a fluid-tight barrier on a surface of theporous implantable member of the present invention.

The size of the particles used in the present invention is one factorused to control the viscosity of the final slurry composition. Notwishing to be bound by a particular theory, it is believed that thesenon-denatured small, uniformly sized particles are critical to theoperation of the present invention. In particular, it is believed thatthe above-referenced properties of these particles enable them tocombine faster and to form stronger, more cohesive sealant compositions,without cross-linking, than previous prior art compositions. Thus,cryogenic milling of the self-aggregating protein followed by sieving ofthe protein comminutate provides the skilled artisan with anunprecedented level of control over the physical properties of the finalslurry composition, including viscosity, protein dispersion andreproducibility. Such control is vital for producing vascularprostheses, such as grafts and endografts, having safe, reliable andconsistent barrier properties.

As set forth hereinabove, the viscosity of the aqueous slurry must becarefully controlled. In particular, the viscosity of the aqueous slurrymust be such that sufficient self-aggregating particles are present toform an effective barrier coating or impregnation to prevent unwantedleakage of, for example, blood, through a porous vascular prosthesistreated therewith. At the same time, however, the aqueous slurry musthave enough flow so that it is easily applied to a porous substrate.Accordingly, viscosities which meet these general limitations may beused in the present invention. Preferably, the aqueous slurrycomposition containing the self-aggregating protein has a viscosity ofbetween about 8,000 centipoise (cps) to about 60,000 cps at 25° C. Morepreferably, the aqueous slurry composition has a viscosity of betweenabout 30,000 cps to about 50,000 cps at 25° C. As discussed previously,the particle size of the protein in the aqueous slurry influences itsviscosity.

Additional parameters, however, are also used to control the viscosityof the slurry composition. For example, the temperature and pH of theslurry composition, the amount of mixing the slurry composition issubjected to, and the final concentration of the self-aggregatingprotein in the final slurry composition are additional factorsinfluencing the final viscosity thereof. The pH of the aqueous slurrymust be monitored in order to ensure that it remains in an aqueousstate. It is within the knowledge of the skilled artisan to select anappropriate pH based on the isoelectric point of the raw material, e.g.,based on the isoelectric point of the self-aggregating protein containedtherein. For example, the pH of the slurry should be maintained no lessthan 0.3 pH units away from the isoelectric point of the raw material.In the case of limed bovine skin type I collagen in which theisoelectric point is 4.2, it is preferred that the pH of the aqueousslurry be maintained in the range of about 3.5 to about 3.9. Desirably,the pH of the slurry should be sufficient to maintain the proteinparticles suspended within the slurry.

As stated hereinabove, the aqueous slurry composition of the presentinvention also contains a biologically acceptable plasticizer forenhancing the flexibility and handling characteristics of theimplantable article. Suitable plasticizers include polyhydric alcoholsincluding for example, glycerol, sorbitol and mannitol. Preferably, theplasticizer accounts for between about 8% to about 30% by weight of theaqueous slurry. In one desired embodiment, the slurry includes about 1to about 3% of self-aggregating protein particles of substantially thesame diameter range; and about 8 to about 30% by weight of theplasticizer, based on the total weight of the slurry.

Additionally, optional agents may also be added to the present aqueousslurry composition. These agents may be used for the purposes ofbioburden control, or to modify the flow characteristics of the slurry.The use of such agents can lead to a device with lower permeabilities.Accordingly, such agents will be referred to hereinafter as“permeability lowering compositions.” An example of such an agent isethanol. Moreover, when reagents like ethanol are used, they confer theadded benefit of functioning as bacteriostatic agents. Preferably, thepermeability lowering composition, if used, accounts for up to about 24%by weight of the aqueous slurry composition.

Bio-active agents may also be added to the present aqueous slurrycomposition. Such agents may be added to the slurry composition toreduce the risk of infection or thrombus formation associated with theimplantation of an implantable article of the present invention.Suitable bio-active agents include, for example, antibiotics,anticoagulants, antibacterial agents and mixtures thereof.

As set forth hereinabove, the aqueous slurry composition of the presentinvention renders the implantable article fluid-tight. For purposes ofthe present invention, “fluid-tight” is intended to mean that the porousimplantable article is rendered essentially non-permeable to liquids,such as for example, blood.

To achieve the desired level of fluid-tightness, the aqueous slurrycomposition of the present invention is placed in intimate contact witha porous implantable article. In the case of a vascular graft, theaqueous slurry composition is placed in intimate contact therewith bycoating or impregnating methods. Such methods include placing theaqueous slurry composition within the graft and forcing it through thepores of the graft with sufficient force to cause the slurry compositionto either coat the surface or penetrate into the pores and intersticesof the graft. The force used to distribute the slurry compositionthrough the porous article may be supplied by pressure means, such asmechanical rollers and the like, or fluidized pressure.

Multiple applications of the present aqueous slurry composition may beapplied to the implantable article. Preferably, between three (3) to six(6) applications of the aqueous slurry composition are applied to theimplantable article. Between each application of the aqueous slurrycomposition, the slurry coated implant is dried. This drying isaccomplished in an oven having a temperature between about 25° C. toabout 35° C. for about 45-75 minutes. In one desired embodiment, animplantable member of the present invention includes a porous substratehaving a surface that includes at least three applications ofself-aggregating protein particles of substantially the same diameter,deposited from the slurry, for forming a fluid-tight seal.

While it is an advantage of the present invention over the prior artthat cross-linking is not required to form an effective fluid barrier, across-linking agent may also be optionally added to the aqueous slurrycomposition, if desired. In such cases, the self-aggregating proteins inthe slurry composition are cross-linked prior to drying of, e.g., theslurry coated vascular graft. Any bio-compatible cross-linking agent maybe used to cross-link the self-aggregating proteins of the presentinvention. Suitable cross-linking agents include, for example,formaldehyde and glutaraldehyde. Preferably, the cross-linking agent ispresent in the aqueous slurry composition from about 0 to about 500parts per million. Alternatively, the cross-linking agent may beintroduced following application of the slurry in either solution orgaseous form.

In another embodiment of the present invention, there is provided animplantable member for use in a body. This implantable member includes aflexible, porous polymeric substrate as previously described. An aqueoussealant composition is in intimate contact with the porous substrate.This sealant composition includes a slurry of a self-aggregating proteincomminutate, a bio-compatible plasticizer and water. Each of thecomponents of this slurry are separately described hereinabove.Furthermore, as set forth above, the viscosity of the sealantcomposition is maintained between about 8,000 cps and about 60,000 cps.Moreover, the pH of the sealant composition is controlled in order tomaintain the protein in suspension in the slurry.

In a preferred embodiment, a porous vascular graft, as hereinbeforedescribed, is coated and/or impregnated with the inventive sealantcomposition to form a fluid-tight barrier thereon.

In a further embodiment of the present invention, there is disclosed aprocess for making a bio-compatible aqueous sealant slurry for renderingimplantable articles fluid-tight. As set forth in more detail below,this process includes the steps of (1) providing a paste containing abio-absorbable self-aggregating protein, (2) milling the paste atcryogenic temperatures to a powder having a uniform particle size, (3)mixing the powder with a plasticizer and water to form theabove-referenced aqueous sealant slurry which has a viscosity of about8,000 centipoise to about 60,000 centipoise at 25° C. and (4)maintaining the slurry at a pH sufficiently outside of the isoelectricpoint of the paste to maintain the self aggregating protein dispersed inthe slurry.

In yet another embodiment of the present invention, there is provided aprocess for preparing a fluid-tight implantable article. This processincludes the steps of (1) providing an aqueous slurry as describedhereinabove, (2) applying the slurry to a surface of a porous, flexiblepolymeric substrate with a force sufficient to ensure close associationof the protein with the porous structure of the substrate, and (3)allowing the slurry to dry.

The following examples are set forth to illustrate the process ofpreparing the slurry and fluid-tight implantable articles of the presentinvention. These examples are provided for purpose of illustration onlyand are not intended to be limiting in any sense.

EXAMPLE 1 Preparation of Self Aggregating Protein Paste

Self aggregating proteins in accordance with the present invention areprepared from the appropriate source, including cell and organ cultures,as well as whole organ explants. In the case of collagen type I and III,fresh calf skins are mechanically stripped from the carcasses of youngcalves, fetuses or stillborns and washed in a rotating vessel with coldrunning water until the water is observed to be free from surface dirt,blood and/or tissues. The subcutis is mechanically cleaned to removecontaminating tissues, such as fat and blood vessels. Subsequently, theskins are cut in the longitudinal direction into strips about 12 cm wideand are placed in a wood or plastic vessel as commonly used in theleather industry.

The skins are dehaired with a flusher solution of IM Ca(OH)2 for 25hours. Alternatively, the skins may be dehaired by mechanical means orby a combination of chemical and mechanical means. Following dehairing,the skins are cut into pieces of approximately 1″×1″ and are washed incold water.

Following washing, 120 kg of the bovine skins are placed in a vesselcontaining 260 L water, 2 L NaOH (50%) and 0.41, H₂O₂ (35%). Thesecomponents are mixed slowly for 12 to 15 hours at 4° C. and are washedwith an excess of tap water for 30 minutes to provide partially purifiedskins. These skins are limed in a solution of 260 L water, 1.21, NaOH(50%) and 1.4 kg CaCO₃ for 5 minutes with slow mixing. This treatment iscontinued twice a day for 25 days. Preferably, this liming processcontinues as described for 0-8 days. Following this treatment, thesolution is decanted and discarded. The skins are then washed with anexcess of tap water for 90 minutes under constant stirring.

The skins are acidified in a solution containing 14 kg HCL (35%) in 70 Lwater with vigorous stirring. The acid is allowed to penetrate into theskins for about 6 hours. Following acidification, the skins are washedin an excess of tap water for about 4 hours or until a pH of about 5 isreached. The pH of the skins is then readjusted to 3.3 to 3.4 usingacetic acid containing 0.5% of a preservative. The purified skins arethen made into a raw paste by grinding in a meat grinder and extrudingthe ground skins under pressure through a series of filter sieves ofconstantly decreasing mesh size. The final product is a white homogenoussmooth paste of pure bovine skin-derived type I collagen. This rawcollagen paste is stored at 0-25° C. until further use.

EXAMPLE 2 Cryogenic Milling Process

Optionally, to accommodate the size of certain milling machinery, 25 kgof the raw paste of Example 1 is made into processable-sized curds byextruding the raw paste through a meat grinder. The meat grinder isoutfitted with an extrusion plate having holes through which the rawpaste is extruded. Preferably, the holes are between about ⅛″ to about⅜″ in diameter.

As the raw paste is extruded, it is allowed to fall into a cryogenicbath containing, for example, liquid nitrogen. When the extrudate hitsthe cryogenic bath, it immediately freezes and takes on a curd-likeshape. These curds are then milled to a powder-like consistency atcryogenic temperatures in, for example, a SPEX 6700 Freezer/Mill (SpeyIndustries, Inc.; Edison, N.J.).

This cryogenically milled powder is passed through a sieve and thesieved material collected. The diameter of each particle in this sievedmaterial is highly uniform. Mesh or screen sizes of the sieving screencan vary between about 0.020 inches to about 0.062 inches (0.5 mm-1.55mm). Such a range in screen size produces uniform particles having adiameter of about 5 gm to about 750 gm, depending upon which screen isused. This powder is preferably stored at temperatures below 0° C. toprevent agglomeration.

EXAMPLE 3 Slurry Preparation and Graft Formation

An aqueous slurry of a bovine type I collagen is prepared according toExample 2 using 1.6% by weight of the collagen powder, 8-30% by weightof glycerin with the balance being water. The viscosity of this slurryis measured at 25° C. Only collagen slurry preparations having aviscosity of between about 8,000 cps and about 60,000 cps are retainedfor further processing. Preferably, the viscosity range of the slurry isbetween about 30,000 cps and about 50,000 cps. Aqueous slurry meetingthis viscosity criteria is then applied to a porous vascular graft underpressure of about 15 psi. This pressure forces the slurry into intimatecontact with the internal porous structure of the graft. The graft isthen dried in an oven at about 25° C. to about 35° C. for between about45 to about 75 minutes. Multiple applications of the slurry may be madeto the graft. Preferably between 3 to 6 applications of the slurry aremade to the graft followed by a drying cycle as described herein betweeneach application. The final graft is then sterilized using gammaradiation.

Optionally, 0-24% of ethanol is added to the slurry to lower thepermeability of the final graft. The collagen particles in the slurryare also optionally cross-linked with 0-500 parts per millionformaldehyde prior to application to the vascular graft. Bio-activeagents are also optionally added to the collagen slurry. Thesebio-active agents include, for example, antibiotics, anticoagulants andantibacterial agents. Preferably, heparin is added to the slurry toincrease the anti-thrombogenicity of the collagen slurry. Grafts made inthis manner have essentially zero porosity, i.e., the grafts have asufficiently low permeability that preclotting is not required prior toimplantation.

EXAMPLE 4 Porosity Tests of Porous Vascular Grafts

The porosity of, e.g., a collagen treated fabric graft of the presentinvention is reduced to less than about 1% after three applications asfollows. A standard water porosity test used to measure water porosityof a graft is as follows. A column of water equivalent to 120 mm Hgpressure is allowed to flow through a 0.5 cm² orifice having a sample ofthe graft over the orifice for one minute. The amount of water collectedin 1 minute is measured and the porosity calculated and expressed asml/min/cmz. Several readings are taken for each sample.

The water porosity of a non-treated Microvel graft fabric (MeadoxMedicals, Inc., Franklin Lakes, N.J.) was about 1,900 ml/min/cm². Theporosity after treating the graft with a composition of the presentinvention is as follows: TABLE I Number of Coatings Porosity 0 1,900 1266 2 146 3 14 4 5 5 2 6 0

In each case the collagen coating is a bovine skin derived-plasticizedslurry prepared in accordance with the composition described in Example3. Based on the results of table I, it is preferable to provide acollagen impregnated graft treated with at least three applications ofthe present slury compositions; and most preferable with four or fiveapplications with drying between each application.

EXAMPLE 5 Preparation of Collagen Type II Slurry Composition

The slurry of Example 3 is made as described with the exception thatcollagen type II is subsituted for collagen type I. The properties ofthe slurry coated graft are substantially identical to Example 3 inflexibility, handling and fluid tightness.

EXAMPLE 6 Preparation of Collagen Type III Slurry Composition

The slurry of Example 3 is made as described with the exception thatcollagen type III is substituted for collagen type I. The properties ofthe slurry coated graft are substantially identical to Example 3 inflexibility, handling and fluid tightness.

EXAMPLE 7 Preparation of Collagen Type IV Slurry Composition

The slurry of Example 3 is made as described with the exception thatcollagen type IV is substituted for collagen type I. The properties ofthe slurry coated graft are substantially identical to Example 3 inflexibility, handling and fluid tightness.

EXAMPLE 8 Preparation of Collagen Type V Slurry Composition

The slurry of Example 3 is made as described with the exception thatcollagen type V is substituted for collagen type I. The properties ofthe slurry coated graft are substantially identical to Example 3 inflexibility, handling and fluid tightness.

EXAMPLE 9 Preparation of Collagen Type VI Slurry Composition

The slurry of Example 3 is made as described with the exception thatcollagen type VI is substituted for collagen type I. The properties ofthe slurry coated graft are substantially identical to Example 3 inflexibility, handling and fluid tightness.

EXAMPLE 10 Preparation of Collagen Type VII Slurry Composition

The slurry of Example 3 is made as described with the exception thatcollagen type VII is substituted for collagen type I. The properties ofthe slurry coated graft are substantially identical to Example 3 inflexibility, handling and fluid-tightness.

EXAMPLE 11 Preparation of Collagen Type VIII Slurry Composition

The slurry of Example 3 is made as described with the exception thatcollagen type VIII is substituted for collagen type I. The properties ofthe slurry coated graft are substantially identical to Example 3 inflexibility, handling and fluid tightness.

EXAMPLE 12 Preparation of Collagen Type IX Slurry Composition

The slurry of Example 3 is made as described with the exception thatcollagen type IX is substituted for collagen type I. The properties ofthe slurry coated graft are substantially identical to Example 3 inflexibility, handling and fluid tightness.

EXAMPLE 13 Preparation of Collagen Type X Slurry Composition

The slurry of Example 3 is made as described with the exception thatcollagen type X is substituted for collagen type I. The properties ofthe slurry coated graft are substantially identical to Example 3 inflexibility, handling and fluid tightness.

EXAMPLE 14 Preparation of Collaien-Type XI Slurry Composition

The slurry of Example 3 is made as described with the exception thatcollagen type XI is substituted for collagen type I. The properties ofthe slurry coated graft are substantially identical to Example 3 inflexibility, handling and fluid tightness.

EXAMPLE 15 Preparation of Collagen Type XII Slurry Composition

The slurry of Example 3 is made as described with the exception thatcollagen type MI is substituted for collagen type I. The properties ofthe collagen coated graft are substantially identical to Example 3 inflexibility, handling and fluid tightness.

EXAMPLE 16 Preparation of Collagen Type XIII Slurry Composition

The slurry of Example 3 is made as described with the exception thatcollagen type XII is substituted for collagen type 1. The properties ofthe slurry coated graft are substantially identical to Example 3 inflexibility, handling and fluid tightness.

EXAMPLE 17 Preparation of Collagen Type XIV Slurry Composition

The slurry of Example 3 is made as described with the exception thatcollagen type XIV is substituted for collagen type 1. The properties ofthe slurry coated graft are substantially identical to Example 3 inflexibility, handling and fluid tightness.

EXAMPLE 18 Preparation of Collagen Type XV Slurry Composition

The slurry of Example 3 is made as described with the exception thatcollagen type XV is substituted for collagen type 1. The properties ofthe slurry coated graft are substantially identical to Example 3 inflexibility, handling and fluid tightness.

EXAMPLE 19 Preparation of Fibronectin Slurry Composition

The slurry of Example 3 is made as described with the exception thatfibronectin is substituted for collagen type 1. The properties of theslurry coated graft are substantially identical to Example 3 inflexibility, handling and fluid tightness.

EXAMPLE 20 Preparation of Vitronectin Slurry Composition

The slurry of Example 3 is made as described with the exception thatvitronectin is substituted for collagen type I. The properties of theslurry coated graft are substantially identical to Example 3 inflexibility, handling and fluid tightness.

EXAMPLE 21 Preparation of Proteoglycan Slurry Composition

The slurry of Example 3 is made as described with the exception that aproteoglycan is substituted for collagen type I. The properties of theslurry coated graft are substantially identical to Example 3 inflexibility, handling and fluid tightness.

EXAMPLE 22 Preparation of Laminin Slurry Composition

The slurry of Example 3 is made as described with the exception thatlaminin is substituted for collagen type I. The properties of the slurrycoated graft are substantially identical to Example 3 in flexibility,handling and fluid tightness.

EXAMPLE 23 Preparation of Hyaluronic Acid Slurry Composition

The slurry of Example 3 is made as described with the exception thathyaluronic acid is substituted for collagen type I. The properties ofthe slurry coated graft are substantially identical to Example 3 inflexibility, handling and fluid tightness.

EXAMPLE 24 Preparation of Tenascin Slurry Composition

The slurry of Example 3 is made as described with the exception thattenascin is substituted for collagen type I. The properties of theslurry coated graft are substantially identical to Example 3 inflexibility, handling and fluid tightness.

EXAMPLE 25 Preparation of Integrin Slurry Composition

The slurry of Example 3 is made as described with the exception that anintegrin is substituted for collagen type I. The properties of theslurry coated graft are substantially identical to Example 3 inflexibility, handling and fluid tightness.

EXAMPLE 26 Preparation of Cadherin Slurry Composition

The slurry of Example 3 is made as described with the exception that acadherin is substituted for collagen type I. The properties of theslurry coated graft are substantially identical to Example 3 inflexibility, handling and fluid tightness.

EXAMPLE 27

In a triangle test, experts in the field where asked to compare thephysical properties of a prior art graft and a graft of the presentinvention. The expert respondents used in this study consisted of 47Thoracic Surgeons (all users of a prior art collagen coated vasculargraft) in attendance at the STS Convention in Palm Springs, Calif., Jan.29 - Feb. 1, 1995. Woven 30 mm vascular grafts were used in this test.

The triangle test included presenting three samples of vascular graftseither simultaneously or successively to each respondent. Two of thegrafts presented to each respondent were always coated with the samecomposition; the third graft was always coated with a differentcomposition. Each respondent was required to pick the sample believed tobe different. After successfully selecting the different sample, therespondent was asked which of the two types of grafts they preferred.

In order to neutralize order and presentatiori bias, both grafts coatedwith a prior art composition and grafts coated with the presentcompositions were used equally often as the different sample (i.e., halfof the respondents were given to evaluate two grafts coated with thepresent composition and one prior art sample and half were given toevaluate two prior art samples and one sample according to the presentinvention). In addition, all samples were presented an equal number oftimes in 1st, 2nd and 3rd positions.

After meeting the above-referenced screening requirements, eachrespondent was given three samples to evaluate for handlingcharacteristics. Respondents were instructed to cut and suture thesamples and to identify which was the different sample. After makingtheir selection, they were then asked to select the preferred sample(s)and provide a reason for their preference. Only those respondents thatcorrectly identified the different sample were included in thepreference analysis.

The results of the triangle study and preference analysis indicated that80% of the experts were able to distinguish between a vascular graftcoated with a prior art composition and a vascular graft coated with acomposition of the present invention. Furthermore, 87% of the expertswho were able to distinguish between the two samples preferred the graftcoated with a composition of the present invention. The followingcharacteristics were cited by the experts as reasons for choosing thegraft coated with composition of the present invention: superiorsoftness (71%); superior flexibility (56%), superior saturability (38%).

By way of summary, as the triangle test and preference analysisdemonstrate, the compositions of the present invention allow for morerepeatable and uniform properties in the 32 final graft product,including better handling properties, i.e., a more consistently obtainedsoft-feel and flexibility. This is in contrast to prior art methodswhich did not use a controlled particle size in the present range, andwhich resulted in a wider degree of variation in the final productincluding variation in the “hand”.

The invention being thus described, it will be obvious that the same maybe varied in many ways. Such variations are not to be regarded as adeparture from the spirit and scope of the invention and, all suchmodifications are intended to be included within the scope of thefollowing claims.

1. An implantable member for use in the body comprising: a porousbiocompatible substrate; the substrate having at least one surfacesealed fluid-tight with self-aggregating protein particles ofsubstantially the same diameter range.
 2. The implantable member ofclaim 1, wherein the self-aggregating protein particles have diametersin the range of about 5 μm to about 750 μm.
 3. The implantable member ofclaim 1, wherein the self-aggregating protein is selected from the groupconsisting of collagen, fibronectin, vitronectin, proteoglycan, laminin,hyaluronic acid, tenascin, integrin, cadherin and mixtures thereof. 4.The implantable member of claim 1, wherein the self-aggregating proteinis collagen.
 5. The implantable member of claim 1, wherein theself-aggregating protein particles are formed from cryogenic milling ofa paste containing the self-aggregating protein.
 6. The implantablemember of claim 1, wherein the self-aggregating protein particles areformed from a deposited aqueous slurry of between about 1 to about 3% ofthe protein particles and about 8 to about 30% plasticizer by weight,based on the total weight of the slurry.
 7. The implantable member ofclaim 6, wherein the pH of the slurry is sufficient to maintain theprotein particles suspended within the slurry.
 8. The implantable memberof claim 6, wherein the plasticizer is a polyhydric alcohol.
 9. Theimplantable member of claim 6, wherein the deposited slurry furtherincludes a bio-active agent.
 10. The implantable member of claim 9,wherein the bio-active agent is selected from the group consisting ofantibiotics, anticoagulants and antibacterial agents.
 11. Theimplantable member of claim 6, wherein the deposited slurry furtherincludes a permeability lowering agent.
 12. The implantable member ofclaim 11, wherein the permeability lowering agent is ethanol.
 13. Theimplantable member of claim 1, wherein the substrate surface includes atleast three applications of the self-aggregating protein particles forforming a fluid-tight seal.
 14. The implantable member of claim 1,wherein the substrate is a porous synthetic vascular graft.
 15. Theimplantable member of claim 14, wherein the vascular graft is made frombiologically compatible fibers or yarns.
 16. The implantable member ofclaim 15, wherein the fibers or yarns are selected from the groupconsisting of polyethylene terephthalate and polytetrafluoroethylene.